With the development of high power diode lasers and the commercialization of an economically viable excitation source in the visible range, the 405 nm diode laser has had a major impact in the biotechnology field, especially in flow cytometry, a laser-based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering. The need for suitable dyes fitting the 405 nm excitation source has inspired researchers towards the development of short wavelength excitable dyes, showing large Stokes Shifts and suitable for bio-conjugation applications.
Coumarin dyes with large Stokes shifts (80 nm or higher) and high fluorescence quantum yield that are efficiently excited by a 405 nm laser source are rare.
Diwu et al in U.S. Pat. No. 8,431,416 describe halogenated 7-hydroxy coumarin dyes that can be used in flow cytometry applications.
Fluorescence of 7-hydroxy coumarins is pH dependent due to the intrinsically high pKa value of the C7-hydroxyl proton.
The protonated or neutral form of these dyes is not fluorescent. For these dyes to become fluorescent, complete deprotonation of the 7-hydroxyl proton is required to give the deprotonated or anionic form. At physiological pH these dyes exist in their neutral form and are not fluorescent and therefore not useful in biological assays.
The incorporation of chlorine atoms in the 6- and/or 8-position of the coumarin core structure lowers the pKa and allows these dyes to be useful near physiological pH. Nevertheless, a strong pH dependency is observed up to pH 6-6.5 and the chlorine atoms may impart a heavy atom effect with consequent reduction of fluorescent quantum yield as well as reduce water solubility. Furthermore, the anionic form of these dyes, required for fluorescence, may be undesirable in some applications as described by Jin et al in ChemBioChem, 2011, 12, 65-70.
On the contrary, 7-amino coumarins are known to be pK independent over a much wider pH range and show excellent photostability and high fluorescence quantum ylelds. The primary amine derivatives such as described by Haugland et al (U.S. Pat. No. 5,696,157) show absorption maxima near the UV (350 nm) and therefor are not suitable in flow cytometry applications with the 405 nm violet excitation source.
Tertiary, or N-di-alkylated, 7-amino coumarins are well known as for example, Alexa Fluor 430 among others. However, most of these dyes are not efficiently excited by a 405 nm laser source. Typical excitation maxima are observed in the 425-450 nm range with emission maxima between 460 and 490 nm.
Introduction of a 3-thienyl substituent into a 7-amino coumarin gives an absorption maximum near 400 nm and an emission maximum near 500 nm. Several other derivatives of 7-amino-3-thienyl coumarins have been described in literature as for example in EurJOC, 2015(3), 496-505 and Dyes and Pigments, 112, 227-235; 2015. These fluorophores have found widespread use in, for example, electroluminescence and dye sensitized solar cells.
However, none of these dyes is water soluble, they are extremely hydrophobic and do not possess a reactive group required for preparing fluorescent conjugates. It is well known that hydrophobic fluorophores tend to show high levels of quenching when conjugated to an analyte.
These quenching effects of the fluorophore may be the result of dye-dye self-quenching or are due to dye-analyte interactions. Self-quenching is caused mainly by hydrophobicity, proximity, high degrees of labeling and fluorophore size. Quenching due to dye-analyte interactions is also associated with the hydrophobicity of the fluorophore, where back folding of the fluorophore towards hydrophobic pockets of the anaiyte results in energy transfer processes between the dye and the hydrophobic side chains of amino acid residues in proteins (e.g. tryptophan and tyrosine).
Therefore, these dyes are not suitable as fluorescent markers in bio-conjugation applications.